NFAT in Immune Response

Antigenic stimulation of lymphocytes and other cells of the immune system initiates a complex series of intracellular signal transduction pathways that lead to the expression of a panel of immunoregulatory genes, whose function is critical to the initiation and coordination of the immune response. NFATs (Nuclear Factors of Activated T-Cells), a family of transcription factors expressed both inside and outside of diverse cell types of the immune system, play a pivotal role in the process. Originally described in T-Cells, NFATs have now been implicated in the activation of mast cells, B-Cells, NK (Natural Killer) cells, monocytes, and play a key role in the expression of a number of immunologically important genes, including a wide array of cytokines: IL-2, IL-3, IL-4, IL-5, IL-13, IFN-Gamma (Interferon-Gamma), TNF-Alpha  (Tumor Necrosis Factor-Alpha), and GMCSF  (Granulocyte-Macrophage Colony-Stimulating Factor), as well as several cell-surface molecules such as CD40L  (CD40 Ligand), FasL  (Fas Ligand) and CTLA4  (Cytotoxic T-Lymphocyte-Associated Antigen-4) (Ref.1 & 2). 

 

NFATs are basically Calcium-dependent transcription factors, activated by stimulation of receptors coupled to Calcium-Calcineurin signals, such as the antigen receptors on T-Cells and B-Cells, Fc-Epsilon receptors on mast cells and basophils, the Fc-Gamma receptors on macrophages and NK cells, the platelet collagen receptors, and receptors coupled to heterotrimeric G-proteins (Ref.2). NFATs serve to couple gene expression to changes in intracellular calcium levels and are regulated primarily at the level of their subcellular localization. In response to antigen receptor signaling, the Calcium-regulated phosphatase Calcineurin acts to directly dephosphorylate NFAT proteins, causing their rapid translocation from the cytoplasm to the nucleus, where they cooperatively bind other transcription factors to induce gene expression. NFAT often functions at composite DNA elements by its interaction with Activating Protein-1 complex (a dimmer of c-Fos  and c-Jun),  GATA4  (GATA Binding Protein-4), and  MEF2  (Mads Box Transcription Enhancer Factor-2), and the subsequent formation of a ternary complex induces expression of NFAT targets (Cytokines and Membrane Proteins).Five members of the NFAT family have been identified in mammals, including four cytoplasmic components: NFATc/NFAT2/NFATc1, NFATp/NFAT1/NFATc2, NFAT3/NFATc4, NFAT4/NFATc3, and the constitutively active nuclear component,  NFAT5  (Ref.4). There are three functional domains in NFAT family proteins: the Rel  similarity domain, which is responsible for the DNA binding activity and interaction with c-Fos  and c-Jun; the NFAT homology region, which regulates the intracellular localization; and the transcriptional activation domain. Combinatorial regulation of gene expression is a powerful mechanism in NFAT signaling that enables tight control of gene expression, via integration of multiple signaling pathways. The four Calcium-regulated transcription factors of the NFAT family act synergistically with c-Fos  and c-Jun  proteins on composite DNA elements which contain adjacent NFAT and c-Fos-c-Jun binding sites, where they form highly stable ternary complexes to regulate the expression of diverse inducible genes. Balanced activation of NFAT and Fos-Jun complex is known to be required for productive immune responses. Concomitant induction of NFAT and Fos-Jun requires concerted activation of two different signaling pathways: Calcium-Calcineurin, which promotes NFAT dephosphorylation, nuclear translocation and activation; and MAPK (Mitogen-Activated Protein Kinase) pathway which promotes the synthesis, phosphorylation and activation of membes of the Fos  and Jun  families of transcription factors, downstream of MAPK pathway (Ref.3 & 5). 

 

Activation of antigen receptors of the immune cells (TCR, BCR, FcRs etc.) and the subsequent stimulation of costimulatory receptors in response to antigen presentation leads to activation of a series of signal transduction events mediated by several cytosolic tyrosine kinases and adaptor proteins like  LAT  (Linker for Activation of T-Cells), SLP76, and GRB2  (Growth Factor Receptor-Bound Protein-2), SLP65  (SH2 Domain Containing Leukocyte Protein-65KD) etc. and various kinases like ITK  (IL-2 Inducible T-cell Kinase), BTK  (Bruton’s Tyrosine Kinase) and  SYK  (Spleen Tyrosine Kinase) (Ref.2). These receptors contain unique cytoplasmic domains essential for downstream signaling, called ITAMs (Immunoreceptor Tyrosine-based Activation Motifs). One critical protein that is recruited to the adaptor proteins upon immunoreceptor stimulation is PLC-Gamma (Phospholipase-C-Gamma) whereas, PLC-Beta (Phospholipase-C-Beta) is activated by the GPCRs (G-Protein Coupled Receptors). PLC is responsible for the production of the second messengers DAG (Diacylglycerol) and IP3 (Inositol Triphosphate) by cleaving  PIP2  (Phosphatidylinositol-4,5-Bisphosphate) at the plasma membrane. DAG activates PKC-Theta  (Protein Kinase-C-Theta), whereas IP3 binds to IP3R  (IP3 Receptor) on the surface of the  ER  (Endoplasmic Reticulum) and releases Ca2+ (Ref.9). This event triggers the opening of CRAC (Ca2+ Release Activated Ca2+ Channels) at the plasma membrane, allowing influx of extracellular Ca2+. The increased Ca2+ levels then activate the protein phosphatase Calcineurin by disrupting the inhibitory effects of Calm (Calmodulin). Calcineurin activation leads to the dephosphorylation of NFAT, allowing it to enter the nucleus, where it cooperates with other transcription factors to bind promoters for the induction of NFAT-mediated gene transcription. Effective phosphate removal by Calcineurin requires its docking on NFAT. Interactions between NFAT and Calcineurin occur at a specific motif in the N-terminus of NFAT, which has the consensus sequence PXIXIT, where X denotes any amino acid. This motif is conserved among different NFAT family members and constitutes the main docking site for Calcineurin on NFAT. NFATs remain in the nucleus while Ca2+ is in elevated concentration and are rapidly phosphorylated and exported to the cytoplasm upon termination of Calcium signaling. The activity of Calcineurin is controlled not only by Calcium and Calmodulin but also by several Calcineurin inhibitors. These include CABIN1 (Calcineurin-Binding Protein-1; also known as CAIN), Cyclosporin-A, AKAP79  (A-Kinase Anchor Protein-79; also known as AKAP5) and members of the MCIP (Modulatory Calcineurin-Interacting Protein) family of Calcineurin inhibitors, which are known as Calcipressins (Ref.2, 3, 4, 5 & 6). 

 

The phosphorylation sites on NFAT are located in three different serine-rich motifs: the SRR1 (Serine-Rich Region-1) motif, and the SPXX (where X denotes any amino acid) repeat motifs: SP2  and SP3. Dephosphorylation of the serine residues in these motifs leads to exposure of the NFAT nuclear localization signal and nuclear import, and it might also control their DNA-binding affinity. Nuclear import of dephosphorylated NFATs is facilitated by Importins. NFAT contains both NLS (Nuclear Localization Sequence) and NES (Nuclear Export Sequence), but one of them is buried in the protein interior, inaccessible to Importin and Exportin. Whether NLS or NES is masked depends on the phosphorylation state of specific serine residues in the regulatory domain.   Phosphorylation of these serine residues exposes NES whereas dephosphorylation exposes NLS. In resting cells, the NLS of the cytoplasmic NFAT is masked due to phosphorylation on these serine residues.  In stimulated cells, an increase of intracellular Calcium ions activates Ccalcineurin to bring about dephosphorylation of NFAT. Consequently, NLS is exposed and NFAT can be carried into the nucleus by the Importin. On the other hand, several kinases phosphorylate NFAT proteins and control their nuclear shuttling, including GSK3 (Glycogen-Synthase Kinase-3), CK1 (Casein Kinase-1), p38 and JNK (c-Jun Kinase). Different kinases (maintenance kinases and export kinases) phosphorylate the various serine-rich motifs in NFAT proteins. Maintenance kinases act in the cytosol to keep NFAT proteins in a fully phosphorylated state and prevent their translocation into the nucleus in resting cells, whereas, export kinases rephosphorylate NFAT in the nucleus and promote its nuclear export, thereby stopping NFAT-mediated transcription after cell stimulation is withdrawn and Calcineurin activity declines. CK1 docks at a conserved motif that is near the N-terminus of NFAT proteins, and it functions as both a maintenance and an export kinase for SRR1. GSK3 functions as an export kinase. In stimulated active cells, it is inhibited by the PI3K-->Akt pathway activated by CD28  costimulation. MAPKs differentially phosphorylate the first serine of SRR1 in the different NFAT proteins: p38 phosphorylates NFAT1, whereas JNK phosphorylates NFAT2. This can potentiate the ability of CK1 to processively phosphorylate the remaining serines in SRR1. Rephosphorylation of NFAT by protein kinases brings about exposure of its NES and can be exported to the cytoplasm by the exportin CRM1  (Required for Chromosome Region Maintenance) (Ref.1, 4 & 7). 

 

The novel PKC isoform, PKC-Theta  is selectively expressed by the integration of TCR and CD28  costimulatory signals. Productive engagement of T-Cells by Antigen Presenting Cells results in recruitment of PKC-Theta  to the T-Cell-Antigen-Presenting Cell contact area--the IS (Immunological Synapse), where it interacts with several signaling molecules like Fyn, Lck  and ZAP70  (Zeta-Chain (TCR) Associated Protein Kinase of 70 kDa) to induce activation signals essential for the activation of transcription factors NF-KappaB, c-Jun  and c-Fos. PKC-Theta  also cooperates with Calcineurin, in transducing signals leading to activation of c-Fos, c-Jun  and NFAT. The MAPK family is known to be involved in inducing IL gene expression through activation of ATF2  (Activating Transcription Factor-2), c-Jun  and c-Fos. IKKs (I-KappaB Kinases) and MAPKs are also recruited by TCR and CD28  costimulatory signals and trigger immune response (Ref.8). 

 

Since members of the NFAT family regulate transcription of genes encoding proteins involved in the induction and/or regulation of the immune response, it is possible that altered transcription activity of NFAT under conditions of its deficit or blockade of expression may account for changes in the immune status of an organism (Ref.3). Janus kinase 3 (Jak3) downstream of the interleukin-7 (IL-7) receptor phosphorylates a single tyrosine residue within the regulatory domain of NFAT2, which induces nuclear translocation and activation of NFAT2 independent of Ca2+ signals and calcineurin in thymocytes. CsA, cyclosporine A; NFAT, nuclear factor of activated T cells. While integration of Ca2+ and other signaling pathways results in productive activation, unopposed Ca2+ signaling leads to tolerance or anergy. Ca2+ -->Calcineurin signaling induces a limited set of anergy-associated genes, distinct from genes induced in the productive immune response and these genes are upregulated in vivo in tolerant T-Cells and are largely NFAT dependent.  NFAT1  induces T-Cell anergy if prevented from interacting with its transcriptional partners: c-Fos  and c-Jun. Thus, in the absence of its transcriptional partners, NFAT imposes a genetic program of lymphocyte anergy that counters the program of productive activation mediated by the cooperative NFAT-Fos-Jun complex. Tolerance is also induced in regulatory T-Cells, in part by producing immunosuppressive cytokines such as TGF-Beta  (Transforming Growth Factor-Beta) and IL-10. Thus, a single transcription factor, NFAT, regulates two contrasting aspects of T-Cell function, mediating nonoverlapping genetic programs of productive activation or anergy depending on the availability of Ca2+ and the presence or absence of its transcriptional partners (Ref.9). Understanding the biochemistry and logic behind these integrative processes will allow development of more selective and efficient pharmaceuticals that suppress, modify, or augment immune responses in autoimmunity, transplantation, allergy, vaccines, and cancer (Ref.10, 11 & 12). As balanced activation of NFAT and Jun-Fos complex is known to be required for productive immune responses, pharmacological interference with the interaction of NFAT with c-Fos  and c-Jun  may be useful in selective manipulation of the immune response (Ref.5). NFAT has also been found to regulate HIV1 (Human Immunodeficiency Virus-1) infection indirectly. In turn, HIV1, and particularly its Tat and Nef gene products, can upregulate NFAT expression and activity. This reciprocal regulation between virus and transcription factor potentially creates a positive feedback loop, which may facilitate the establishment of early HIV1 infection and, later, the transition from latent to productive infection. The immunosuppressive drug Cyclosporin-A inhibits NFAT activity and represents a potential treatment for HIV1 infection (Ref.13).